RNA Sequencing of Human Peripheral Nerve in Response to Injury: Distinctive Analysis of the Nerve Repair Pathways

Abstract

The development of regenerative therapies for central nervous system diseases can likely benefit from an understanding of the peripheral nervous system repair process, particularly in identifying potential gene pathways involved in human nerve repair. This study employed RNA sequencing (RNA-seq) technology to analyze the whole transcriptome profile of the human peripheral nerve in response to an injury. The distal sural nerve was exposed, completely transected, and a 1 to 2 cm section of nerve fascicles was collected for RNA-seq from six participants with Parkinson's disease, ranging in age between 53 and 70 yr. Two weeks after the initial injury, another section of the nerve fascicles of the distal and pre-degenerated stump of the nerve was dissected and processed for RNA-seq studies. An initial analysis between the pre-lesion status and the postinjury gene expression revealed 3,641 genes that were significantly differentially expressed. In addition, the results support a clear transdifferentiation process that occurred by the end of the 2-wk postinjury. Gene ontology (GO) and hierarchical clustering were used to identify the major signaling pathways affected by the injury. In contrast to previous nonclinical studies, important changes were observed in molecular pathways related to antiapoptotic signaling, neurotrophic factor processes, cell motility, and immune cell chemotactic signaling. The results of our current study provide new insights regarding the essential interactions of different molecular pathways that drive neuronal repair and axonal regeneration in humans.

Keywords: RNA-seq; Schwann cell; Wallerian degeneration; graft; neurodegenerative diseases; peripheral nerve.

Conflict of interest statement

Declaration of Conflicting Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Figures

Fig. 1.

Photographs of the peripheral nerve fascicles analyzed in this study. (A) The samples collected in Stage I of the DBS surgery (pre-lesion sample). (B) The samples collected in Stage II of the DBS surgery (post-lesion sample). DBS: deep brain stimulation.

Fig. 2.

(A) Correlation matrix or Pearson’s r for the transcriptional profile of every subject vs every other subject. Scale bar: Correlation values range from 0.4 (blue—less similar) to 1 (red—more similar). This visualization shows strong agreement among different profiles within each stage, and a sharp distinction between stages. (B) Differences between Stage 1 and Stage 2. Log 2 scale fold changes (x-axis) are plotted as a function of P-value (inverted log 10 scale—volcano plot). Results that exceed conservative q-value (q ≤ 0.0003) and fold change (|FC| ≥ 4) cutoffs are highlighted (blue—downregulated in Stage 2, red—upregulated in Stage 2). FC: fold change.

Fig. 3.

Heat map showing all significantly differentially expressed (q < 0.05, |FC| > 2) gene transcripts annotated with the GO term “Growth factor Activity” (GO:0008083). Genes are organized by Ward hierarchical clustering. Dendrograms are scaled to hierarchical clustering distance; longer branches represent more distant clusters. FC: fold change; GO: gene ontology.

Fig. 4.

Heat map showing all significantly differentially expressed (q < 0.05, |FC| > 2) gene transcripts annotated with the GO term “Myelination” (GO:0042552). Genes are organized by Ward hierarchical clustering. Dendrograms are scaled to hierarchical clustering distance; longer branches represent more distant clusters. FC: fold change; GO: gene ontology.

Fig. 5.

Heat map showing all significantly differentially expressed (q < 0.05, |FC| > 2) gene transcripts annotated with the GO term “Epithelial–Mesenchymal Transition” (GO:0001837) or “Schwann Cell Differentiation” (GO:0014037). Genes are organized by Ward hierarchical clustering. Dendrograms are scaled to hierarchical clustering distance; longer branches represent more distant clusters. FC: fold change; GO: gene ontology.

Fig. 6.

Heat map showing all significantly differentially expressed (q < 0.05, |FC| > 2) gene transcripts annotated with the GO term “Negative Regulation of Apoptotic Processes” (GO:0043066) or “Negative Regulation of Neuron Death (GO:1901215).” Genes are organized by Ward hierarchical clustering. Dendrograms are scaled to hierarchical clustering distance; longer branches represent more distant clusters. FC: fold change; GO: gene ontology.